Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin
Liat Linde, … , Eitan Kerem, Batsheva Kerem
Liat Linde, … , Eitan Kerem, Batsheva Kerem
Published March 1, 2007
Citation Information: J Clin Invest. 2007;117(3):683-692. https://doi.org/10.1172/JCI28523.
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Research Article Pulmonology

Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin

Abstract

Aminoglycosides can readthrough premature termination codons (PTCs), permitting translation of full-length proteins. Previously we have found variable efficiency of readthrough in response to the aminoglycoside gentamicin among cystic fibrosis (CF) patients, all carrying the W1282X nonsense mutation. Here we demonstrate that there are patients in whom the level of CF transmembrane conductance regulator (CFTR) nonsense transcripts is markedly reduced, while in others it is significantly higher. Response to gentamicin was found only in patients with the higher level. We further investigated the possibility that the nonsense-mediated mRNA decay (NMD) might vary among cells and hence governs the level of nonsense transcripts available for readthrough. Our results demonstrate differences in NMD efficiency of CFTR transcripts carrying the W1282X mutation among different epithelial cell lines derived from the same tissue. Variability was also found for 5 physiologic NMD substrates, RPL3, SC35 1.6 kb, SC35 1.7 kb, ASNS, and CARS. Importantly, our results demonstrate the existence of cells in which NMD of all transcripts was efficient and others in which the NMD was less efficient. Downregulation of NMD in cells carrying the W1282X mutation increased the level of CFTR nonsense transcripts and enhanced the CFTR chloride channel activity in response to gentamicin. Together our results suggest that the efficiency of NMD might vary and hence have an important role in governing the response to treatments aiming to promote readthrough of PTCs in many genetic diseases.

Authors

Liat Linde, Stephanie Boelz, Malka Nissim-Rafinia, Yifat S. Oren, Michael Wilschanski, Yasmin Yaacov, Dov Virgilis, Gabriele Neu-Yilik, Andreas E. Kulozik, Eitan Kerem, Batsheva Kerem

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Figure 2

Effect of NMD inhibition on the level of endogenous CFTR transcripts.

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Effect of NMD inhibition on the level of endogenous CFTR transcripts. ...
(A) Effect of indirect NMD inhibition by CHX on the level of CFTR transcripts. The level was measured by real-time PCR and normalized to that of RPS9 and the ratio between these normalized levels following treatment was calculated and compared to the ratio in untreated cells. (B) Western blot analysis in CFP15a (upper panels) and CFP15b (lower panel) cells following sequence-specific downregulation of UPF1 and UPF2. (C and D) Effect of direct NMD inhibition by siRNA directed against UPF1 (C) and UPF2 (D) on the level of CFTR transcript. The level was measured and normalized as described in A. The increase in the level of CFTR transcripts is shown as mean ± SEM.